The technique of radioimmunoassay (RIA) because of the disadvantages, such as radioactive contamination, short half-lives, short shelf-life, and so on, will be abandoned. The sensitivity and reproducibility of enzyme-linked immunosorbent assay (ELISA) are not better than those of radioimmunoassay, and the enzymatic activity and stability of chromogenic substrates remain to be improved. Chemiluminescence immunoassay (CLIA) because of short-lived light emission and unrepeatable detection, susceptibility to environmental interference and expensive reagents, cannot be widely used.
By contrast, time-resolved immunoassay (TRFIA) with high sensitivity, low background, good stability and wide linear ranges now has been widely recognized as the most promising nonradioactive labeled immunoassay. In recent years, the analysis systems have been developed described as follows:
Dissociation-enhanced lanthanide time-resolved fluoroimmunoassay (DELFIA): the most widely used, consists of a tracer, a bifunctional chelating agent and an enhancer. Fluorescence enhancement is one of the important factors to achieve extremely high sensitivity. Lanthanide ions forming microcapsules with an enhancer solution can effectively prevent luminescence-quenching by water molecules, thus greatly enhancing the fluorescence intensity of the system. In addition, the bifunctional chelating agent is also one of the key factors, and a labeling ratio between europium ion as the first selected label and protein is between 10 and 20. But the bifunctional chelating agent as the label issusceptible to interference caused by external substances such as exogenous rare earth ions, ethylenediaminetetraacetic acid, heparin and other anticoagulants. The test sample must be serum, which has very strict operational requirements and is rather expensive.
Solid-phase time-resolved fluoroimmunoassay (FIAgen) is a time-resolved immunoassay using bifunctional rare earth chelating agents as labels. It has an advantage that fluorescence can be directly detected without the need for adding an enhancer solution, but its sensitivity of the detection is much lower than DELFIA.
Moreover, fluorescent nanoparticles coated with rare earth chelates are also used in time-resolved immunoassay. Each nanoparticle contains thousands of rare earth chelates. Therefore, the detection sensitivity is greatly improved, but it is affected by instability of rare earth chelates, such as easy leaking, susceptibility to photobleaching, and so on.
It should be mentioned that rare earth nanomaterials have advantages such as stable property, large specific surface area, strong modifiability, low synthetic cost, and so on, and have recently been generally recognized as a promising and new generation of fluorescent labels for biological materials. However, the luminescence of rare earth nanomaterials is via absorption of light and sensitized luminescence through the intra-4f configuration transitions within the rare earth ions, which gives a low molar extinction coefficient and leads to weak luminescence. Hence, when rare earth nanomaterials are used as labels for direct detection, their applications are limited by the low sensitivity.